Small molecule printing

ABSTRACT

The present invention provides compositions and methods to facilitate the identification of compounds that are capable of interacting with a biological macromolecule of interest. In one aspect, a composition is provided that comprises an array of one or more types of chemical compounds attached to a solid support, wherein the density of the array of compounds is at least 1000 spots per cm 2 . These compounds are typically attached to the solid support through a covalent interaction. In another aspect, the present invention provides methods for utilizing these arrays to identify small molecule partners for biological macromolecules of interest.

RELATED APPLICATIONS

The present application is a continuation of and claims priority under35 U.S.C. §120 to U.S. patent application, U.S. Ser. No. 10/998,867,filed Nov. 29, 2004, which is a divisional of and claims priority under35 U.S.C. §120 to U.S. patent application, U.S. Ser. No. 09/567,910,filed May 10, 2000, which claims priority under 35 U.S.C. §119(e) toU.S. provisional application, U.S. Ser. No. 60/133,595, filed May 11,1999; each of which is incorporated herein by reference.

BACKGROUND OF INVENTION

The ability to identify small molecule ligands for any protein ofinterest has far-reaching implications, both for the elucidation ofprotein function and for the development of novel pharmaceuticals. Withthe introduction of split-pool strategies for synthesis (Furka et al.,Int. J. Pept. Protein Res. 1991, 37, 487; Lam et al., Nature 1991, 354,82; each of which is incorporated herein by reference) and thedevelopment of appropriate tagging technologies (Nestler et al., J. Org.Chem. 1994, 59, 4723; incorporated herein by reference), chemists arenow able to prepare large collections of natural product-like compoundsimmobilized on polymeric synthesis beads (Tan et al., J. Am. Chem. Soc.1998, 120, 8565; incorporated herein by reference). These librariesprovide a rich source of molecules for the discovery of new proteinligands.

With such libraries in hand, the availability of efficient methods forscreening these compounds becomes imperative. One method that has beenused extensively is the on-bead binding assay (Lam et al., Chem. Rev.1997, 97, 411; incorporated herein by reference). An appropriatelytagged protein of interest is mixed with the library and beads displayedcognate ligands are subsequently identified by a chromagenic orflorescence-linked assay (Kapoor et al., J. Am. Chem. Soc. 1998, 120,23; Morken et al., J. Am. Chem. Soc. 1998, 120, 30; St. Hilare et al.,J. Am. Chem. Soc. 1998, 120, 13312; incorporated herein by reference).Despite the proven utility of this approach, it is limited by the smallnumber of proteins that can be screened efficiently. In principle, thebeads can be stripped of one protein and reprobed with another; however,this serial process is slow and limited to only a few iterations. Inorder to identify a specific small molecule ligand for every protein ina cell, tissue or organism, high-throughput assays that enable eachcompound to be screened against many different proteins in a parallelfashion are required. Although Brown et al. (U.S. Pat. No. 5,807,522;incorporated herein by reference) have developed an apparatus and amethod for forming high density arrays of biological macromolecules forlarge scale hybridization assays in numerous genetic applications,including genetic and physical mapping of genomes, monitoring of geneexpression, DNA sequencing, genetic diagnosis, genotyping of organisms,and distribution of DNA reagents to researchers, the development of ahigh density array of natural product-like compounds for high-throughputscreening has not been achieved.

Clearly, it would be desirable to develop methods for generating highdensity arrays that would enable the screening of compounds present inincreasingly complex natural product-like combinatorial libraries in ahigh-throughput fashion to identify small molecule partners forbiological macromolecules of interest.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods to facilitatethe high-throughput screening of compounds for the identification ofdesirable properties or interactions. In a preferred embodiment, thepresent invention provides compositions and methods to facilitate theidentification of compounds that are capable of interacting with abiological macromolecule of interest. In one aspect, a composition isprovided that comprises an array of more than one type of chemicalcompounds attached to a solid support, wherein the density of the arrayof compounds comprises at least 1000 spots per cm², more preferably atleast 5000 spots per cm², and most preferably at least 10,000 spots percm². In another aspect, a composition is provided that comprises aplurality of one or more types of non-oligomeric chemical compoundsattached to a glass or polymer support, wherein the density of the arrayof compounds comprises at least 1000 spots per cm². In a particularlypreferred embodiment, the chemical compounds are non-peptidic andnon-oligomeric. In particularly preferred embodiments, these compoundsare attached to the solid support through a covalent interaction. Inanother particularly preferred embodiment, small molecules are attachedto the solid support through a covalent interaction. In a particularlypreferred embodiment, the compounds are attached to the solid supportusing a Michael addition reaction. In another preferred embodiment, thecompounds are attached to the solid support using a silylation reaction.In general, these inventive arrays are generated by: (1) providing asolid support, wherein said solid support is functionalized with aselected chemical moiety capable of interacting with a desired chemicalcompound to form an attachment; (2) providing one or more solutions ofone or more types of compounds to be attached to the solid support; and(3) delivering said one or more types of compounds to the solid support,whereby an array of compounds is generated and the array comprises adensity of at least 1000 spots per cm² (FIG. 1). In other embodiments,the array comprises a density of at least 5000 spots per cm², and morepreferably at least 10,000 spots per cm².

In another aspect, the present invention provides methods for utilizingthese arrays to identify small molecule partners for biologicalmacromolecules (e.g., proteins, peptides, polynucleotides) of interestcomprising: (1) providing an array of one or more types of compounds(e.g., more preferably, small molecules), wherein the array has adensity comprising at least 1000 spots per cm²; (2) contacting the arraywith one or more types of biological macromolecules of interest; and (3)determining the interaction of specific small molecule-biologicalmacromolecule partners (FIG. 1). In particularly preferred embodiments,the biological macromolecules of interest comprise a collection of oneor more recombinant proteins. In another preferred embodiment, thebiological macromolecules of interest comprise a collection ofmacromolecules from a cell lysate. In another preferred embodiment, thebiological macromolecules of interest comprise a polynucleotide.

DEFINITIONS

Unless indicated otherwise, the terms defined below have the followingmeanings:

“Antiligand”: As used herein, the term “antiligand” refers to theopposite member of a ligand/anti-ligand binding pair. The anti-ligandmay be, for example, a protein or other macromolecule receptor in aneffector/receptor binding pair.

“Compound”: The term “compound” or “chemical compound” as used hereincan include organometallic compounds, organic compounds, metals,transitional metal complexes, and small molecules. In certain preferredembodiments, polynucleotides are excluded from the definition ofcompounds. In other preferred embodiments, polynucleotides and peptidesare excluded from the definition of compounds. In a particularlypreferred embodiment, the term compounds refers to small molecules(e.g., preferably, non-peptidic and non-oligomeric) and excludespeptides, polynucleotides, transition metal complexes, metals, andorganometallic compounds.

“Ligand”: As used herein, the term “ligand” refers to one member of aligand/anti-ligand binding pair, and is referred to herein also as“small molecule”. The ligand or small molecule may be, for example, aneffector molecule in an effector/receptor binding pair.

“Michael Addition”: The term “Michael addition” refers to the reactionin which compounds containing electron-rich groups (e.g., groupscontaining sulfur, nitrogen, oxygen, or a carbanion) add, in thepresence of base, to olefins of the from C═C—Z (including quinones),where Z is an electron-withdrawing group, such as aldehydes, ketones,esters, amides, nitriles, NO₂, SOR, SO₂R, etc.

“Microarray”: As used herein, the term “microarray” is a regular arrayof regions, preferably spots of small molecule compounds, having adensity of discrete regions of at least about 1000/cm².

“Natural Product-Like Compound”: As used herein, the term “naturalproduct-like compound” refers to compounds that are similar to complexnatural products which nature has selected through evolution. Typically,these compounds contain one or more stereocenters, a high density anddiversity of functionality, and a diverse selection of atoms within onestructure. In this context, diversity of functionality can be defined asvarying the topology, charge, size, hydrophilicity, hydrophobicity, andreactivity to name a few, of the functional groups present in thecompounds. The term, “high density of functionality”, as used herein,can preferably be used to define any molecule that contains preferablythree or more latent or active diversifiable functional moieties. Thesestructural characteristics may additionally render the inventivecompounds functionally reminiscent of complex natural products, in thatthey may interact specifically with a particular biological receptor,and thus may also be functionally natural product-like.

“Peptide”: According to the present invention, a “peptide” comprises astring of at least three amino acids linked together by peptide bonds.Peptide may refer to an individual peptide or a collection of peptides.Inventive peptides preferably contain only natural amino acids, althoughnon-natural amino acids (i.e., compounds that do not occur in nature butthat can be incorporated into a polypeptide chain; see, for example,http://www.cco.caltech.edu/˜dadgrp/Unnatstruct.gif, which displaysstructures of non-natural amino acids that have been successfullyincorporated into functional ion channels) and/or amino acid analogs asare known in the art may alternatively be employed. Also, one or more ofthe amino acids in an inventive peptide may be modified, for example, bythe addition of a chemical entity such as a carbohydrate group, aphosphate group, a farnesyl group, an isofarnesyl group, a fatty acidgroup, a linker for conjugation, functionalization, or othermodification, etc.

“Polynucleotide” or “oligonucleotide”: Polynucleotide or oligonucleotiderefers to a polymer of nucleotides. The polymer may include naturalnucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine,deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine),nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine,pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine,C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine,C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine,7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine,O(6)-methylguanine, and 2-thiocytidine), chemically modified bases,biologically modified bases (e.g., methylated bases), intercalatedbases, modified sugars (e.g., 2′-fluororibose, ribose, 2′-deoxyribose,arabinose, and hexose), or modified phosphate groups (e.g.,phosphorothioates and 5′-N-phosphoramidite linkages).

“Small Molecule”: As used herein, the term “small molecule” refers to anon-peptidic, non-oligomeric organic compound either synthesized in thelaboratory or found in nature. Small molecules, as used herein, canrefer to compounds that are “natural product-like”, however, the term“small molecule” is not limited to “natural product-like” compounds.Rather, a small molecule is typically characterized in that it containsseveral carbon-carbon bonds, and has a molecular weight of less than1500, although this characterization is not intended to be limiting forthe purposes of the present invention. Examples of “small molecules”that occur in nature include, but are not limited to, taxol, dynemicin,and rapamycin. Examples of “small molecules” that are synthesized in thelaboratory include, but are not limited to, compounds described in Tanet al., (“Stereoselective Synthesis of over Two Million Compounds HavingStructural Features Both Reminiscent of Natural Products and Compatiblewith Miniaturized Cell-Based Assays” J. Am. Chem. Soc. 1998, 120, 8565)and pending application Ser. No. 08/951,930 “Synthesis of CombinatorialLibraries of Compounds Reminiscent of Natural Products”, the entirecontents of which are incorporated herein by reference. In certain otherpreferred embodiments, natural-product-like small molecules areutilized.

DESCRIPTION OF THE DRAWING

FIG. 1 depicts one preferred embodiment of the complete process of smallprinting and assaying for chemical compounds with desired properties.The process begins with the combinatorial library. The library istransferred to stock plates which are used to print the compounds ontoglass slides. The slide is then used to assay for chemical compoundswith the desired property.

FIG. 2 depicts the preparation of maleimide-derivatized glass slides.

FIG. 3 shows the attachment of phenolic hydroxyl groups using aMitsunobu activation of the glass surface.

FIG. 4 shows the attachment of compounds having a secondary alcohol to asilicon tetrachloride-activated glass surface.

FIG. 5 shows other attachment chemistries which may be used in smallmolecule printing.

FIG. 6 depicts test compounds used to demonstrate the concept of smallmolecule printing.

FIG. 7 depicts small molecules printed on maleimide-derivatized glassslides and detected with fluorophore-conjugated proteins. Compounds wereprinted according to the pattern illustrated in panel (D). Yellowcircles indicate thiol-derivatized small molecule. (A) indicates a slidedetected with Cy5-streptavidin. (B) indicates a slide detected withDI-22 followed by Cy5-goat-anti-mouse antibody. (C) indicates a slidedetected with RGS (His)₆-FKBP12 followed by mouse-anti-RGS (His)₆antibody followed by Cy5-goat-anti-mouse antibody.

FIG. 8 depicts small molecules printed on a maleimide-derivatized glassslide and detected with FITC-streptavidin (blue), Cy3-DI-22 (green), andCy5-FKBP12 (red). The full slide contains 10,800 distinct spots and wasprepared using only one bead for each of the three small moleculesprinted (1a, 2a, and 3a as shown in FIG. 6).

FIG. 9 shows the activation of glass slides for the covalent attachmentof alcohols.

FIG. 10 shows a) alcohols attached to 500-560 μm polystyrene resinthrough a silyl-containing linker; b-e) a nine spot microarray printedaccording to the pattern in 6 f and visualized in the followingchannels: b) Cy5 (false-colored red), c) Cy3 (false-colored green), d)FITC (false-colored purple), e) Cy5, Cy3, and FITC. Average distancebetween spots=400 μm; average spot diameter=300 μm.

FIG. 11 shows a microarray of primary, secondary, phenolic, and methylester derivatives of an FKBP ligand. Slides were probed with Cy5-labeledFKBP (false-colored red).

FIG. 12 shows a) the general structure of a small-molecule library, 78members of which were placed in the wells of a 96-well plate; b) thestructure of two additional ‘tagged’ library members; c) alcoholmicroarray onto which 78 members of the small molecule library and twotagged members were printed. Protein binding detected with Cy5-FKBP(false-colored red) and FITC-streptavidin (false-colored green).

FIG. 13 shows the master template used to fabricate custom slide-sizedreaction vessels that enable the uniform application of ˜1.4 mL solutionto one face of a 2.5 cm×7.5 cm slide.

FIG. 14 shows the method of making the slide-sized reaction vessels.

FIG. 15 shows the application of reagent to one surface of a slide.

FIG. 16 shows the microarraying robot used to create the small moleculearrays.

FIG. 17 shows the print head of the robot.

FIG. 18 shows the array pin of the robot.

DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

As discussed above, the recent advances in the generation of complexchemical libraries of natural product-like compounds having as many as,or more than, one million members, has led to the subsequent need tofacilitate the efficient screening of these compounds for biologicalactivity. Towards this end, the present invention provides methods andcompositions to enable the high-throughput screening of very largenumbers of chemical compounds to identify those with desirableproperties of interest. In preferred embodiments, methods andcompositions are provided to enable the high-throughput screen of verylarge numbers of chemical compounds to identify those compounds capableof interacting with biological macromolecules.

In one aspect, the present invention provides compositions comprisingarrays of chemical compounds, attached to a solid support having adensity of at least 1000 spots per cm², and methods for generating thesearrays. In particularly preferred embodiments, the present inventionprovides arrays of small molecules, more preferably natural product-likecompounds, that are generated from split-and-pool synthesis techniques,parallel synthesis techniques, and traditional one-at-a time synthesistechniques. Additionally, existing collections of compounds may also beutilized in the present invention, to provide high density arrays thatcan be screened for desirable characteristics. In another aspect, thepresent invention provides methods for the identification of ligand(small molecule)-antiligand (biological macromolecule) binding pairsusing the chemical compound arrays. It is particularly preferred thatthe antiligands comprise recombinant protein, and it is moreparticularly preferred that a library of recombinant proteins isutilized in the detection method. In another preferred embodiment, theantiligands comprise macromolecules from cell lysates.

Small Molecule Printing

As discussed above, in one aspect, the present invention providesmethods, referred to herein as “small molecule printing”, for thegeneration of high density arrays and the resulting compositions.According to the method of the present invention, a collection ofchemical compounds, or one type of compound, can be “printed” onto asupport to generate extremely high density arrays. In general, thismethod comprises (1) providing a solid support, wherein the solidsupport is functionalized with a selected chemical moiety capable ofinteracting with a desired chemical compound to form an attachment; (2)providing one or more solutions of the same or different chemicalcompounds to be attached to the solid support; and (3) delivering theone or more solutions of the same or different chemical compounds to thesolid support, whereby an array of compounds is generated and the arrayhas a density of at least 1000 spots per cm².

As one of ordinary skill in the art will realize, although any desiredchemical compound capable of forming an attachment with the solidsupport may be utilized, it is particularly preferred that naturalproduct-like compounds, preferably small molecules, generated fromsplit-and-pool library or parallel syntheses are utilized. Examples oflibraries of natural product-like compounds that can be utilized in thepresent invention include, but are not limited to shikimic acid-basedlibraries, as described in Tan et al. (“Stereoselective Synthesis ofover Two Million Compounds Having Structural Features Both Reminiscentof Natural Products and Compatible with Miniaturized Cell-Based Assays”,J. Am. Chem. Soc., 1998, 120, 8565) and incorporated herein byreference. As will be appreciated by one of ordinary skill in the art,the use of split-and-pool libraries enables the more efficientgeneration and screening of compounds. However, small moleculessynthesized by parallel synthesis methods and by traditional methods(one-at-a-time synthesis and modifications of these structures) can alsobe utilized in the compositions and methods of the present invention, ascan naturally occurring compounds, or other collections of compounds,preferably non-oligomeric compounds, that are capable of attaching to asolid support without further synthetic modification.

As will be realized by one of ordinary skill in the art, insplit-and-pool techniques (see, for example, Furka et al., Abstr. 14thInt. Congr. Biochem., Prague, Czechoslovakia, 1988, 5, 47; Furka et al.,Int. J. Pept. Protein Res. 1991, 37, 487; Sebestyen et al., Bioorg. Med.Chem. Lett. 1993, 3, 413; each of which is incorporated herein byreference), a mixture of related compounds can be made in the samereaction vessel, thus substantially reducing the number of containersrequired for the synthesis of very large libraries, such as thosecontaining as many as or more than one million library members. As anexample, a solid support bound scaffold can be divided into n vessels,where n represents the number of species of reagent A to be reacted withthe support bound scaffold. After reaction, the contents from n vesselsare combined and then split into m vessels, where m represents thenumber of species of reagent B to be reacted with the support boundscaffold. This procedure is repeated until the desired number ofreagents are reacted with the scaffold structures to yield a desiredlibrary of compounds.

As mentioned above, the use of parallel synthesis methods are alsoapplicable. Parallel synthesis techniques traditionally involve theseparate assembly of products in their own reaction vessels. Forexample, a microtiter plate containing n rows and m columns of tinywells which are capable of holding a small volume of solvent in whichthe reaction can occur, can be utilized. Thus, n variants of reactanttype A can be reacted with m variants of reactant type B to obtain alibrary of n×m compounds.

Subsequently, once the desired compounds have been provided using anappropriate method, solutions of the desired compounds are prepared. Ina preferred embodiment, compounds are synthesized on a solid support andthe resulting synthesis beads are subsequently distributed intopolypropylene microtiter plates at a density of one bead per well. Inbut one example, as discussed below in the Examples, the attachedcompounds are then released from their beads and dissolved in a smallvolume of suitable solvent. Due to the minute quantities of compoundpresent on each bead, extreme miniaturization of the subsequent assay isrequired. Thus, in a particularly preferred embodiment, a high-precisiontranscription array robot (Schena et al., Science 1995, 270, 467; Shalonet al., Genome Research 1996, 6, 639; each of which is incorporatedherein by reference) can be used to pick up a small volume of dissolvedcompound from each well and repetitively deliver approximately 1 mL ofsolution to defined locations on a series of chemically-derivatizedglass microscope slides. These chemically-derivatized glass microscopeslides are preferably prepared using custom slide-sized reaction vesselsthat enable the uniform application of solution to one face of the slideas shown and discussed in the Examples. This results in the formation ofmicroscopic spots of compounds on the slides and in preferredembodiments these spots are 200-250 μm in diameter. It will beappreciated by one of ordinary skill in the art, however, that thecurrent invention is not limited to the delivery of 1 nL volumes ofsolution and that alternative means of delivery can be used that arecapable of delivering picoliter or smaller volumes. Hence, in additionto a high precision transcription array robot, other means fordelivering the compounds can be used, including, but not limited to, inkjet printers, piezoelectric printers, and small volume pipetting robots.

As discussed, each compound contains a common functional group thatmediates attachment to a support surface. It is preferred that theattachment formed is robust and therefore the formation of covalentattachments are particularly preferred. A variety of chemical linkagescan be employed to generate the high density arrays of chemicalcompounds. In addition to the robustness of the linkage, otherconsiderations include the solid support to be utilized and the specificclass of compounds to be attached to the support. Particularly preferredsupports include, but are not limited to glass slides, polymer supportsor other solid-material supports, and flexible membrane supports.

In but one example, and as discussed in Example 1, a Michael addition(March, Advanced Organic Chemistry (4th ed.), New York: John Wiley &Sons, 1992, 795-797; incorporated herein by reference) can be employedto attach compounds to glass slides. In one embodiment, as shown in FIG.2, plain glass slides are derivatized to give surfaces that are denselyfunctionalized with maleimide groups. Compounds containing thiol groupscan then be provided. These thiol-containing compounds readily attach tothe surface upon printing via the expected thioether linkage. As one ofordinary skill in the art will realize, other nucleophilic S-, N-, andO-containing compounds can be generated to facilitate attachment of thechemical compound to the solid support via Michael addition, asdescribed above. Other electrophilic Michael acceptors can also beutilized; however, maleimides and vinyl sulfones are particularlypreferred because the hydrophilicity of these groups is believed to playa role in the observed lack of nonspecific protein binding to the slidesurface in aqueous buffer.

In another example, and as discussed in Example 2, a silylation reactioncan be employed to attach compounds to a glass slide. Plain glass slidesare derivatized to yield surfaces that are densely functionalized withsilyl halides. Compounds containing hydroxyl groups can then be providedand contacted with the functionalized glass surface. The hydroxylcontaining compounds readily attach to the surface through thesilicon-oxygen bond formed by nucleophilic substitution on the silylhalide. In a preferred embodiment, the silyl halide is silyl chloride,bromide, or iodide. In other preferred embodiments, leaving groups onthe silicon such as mesylate and tosylate are used rather than halides.Preferably, the hydroxyl groups of the compounds to be attached areunhindered (e.g., primary alcohols).

In another preferred embodiment, compounds with phenolic hydroxyl groupsare attached to a glass surface using Mitsunobu activation of thesurface as shown in FIG. 3 (Derrick et al., Tetrahedron Lett. 1991, 32,7159; incorporated herein by reference). In yet another preferredembodiment, compounds with secondary alcohols are attached a glasssurface activated with silicon tetrachloride (FIG. 4).

Other linkages (FIG. 5) that can be employed in the preparation of theinventive arrays include, but are not limited to disulfide bonds, amidebonds, ester bonds, ether bonds, hydrazone linkages, carbon-carbonbonds, metal ion complexes, and noncovalent linkages mediated by, forexample, hydrophobic interactions or hydrogen bonding. In certainpreferred embodiments, coupling of acids and amines, coupling ofaldehydes and hydrazide, coupling of trichlorocyanuric acid and amines,addition of amines to quinones, attachment of thiols to mercury,addition of sulfhydryls, amines, and hydroxyls to open bis-epoxides,photoreactions of azido compounds to give insertions via a nitreneintermediate, or coupling of diols to boronate is used in thepreparation of the inventive arrays. It will be appreciated by one ofskill in this art that the specific linkages to be utilized should beselected to be (1) robust enough so that the small molecules are notinadvertently cleaved during subsequent assaying steps, and (2) inert sothat the functionalities employed do not interfere with the subsequentassaying steps.

Methods for Detecting Biological Activity

It will be appreciated by one of ordinary skill in the art that thegeneration of arrays of compounds having extremely high spatialdensities facilitates the detection of binding and/or activation eventsoccurring between compounds in a specific chemical library andbiological macromolecules. Thus, the present invention provides, in yetanother aspect, a method for identifying small molecule partners forbiological macromolecules of interest. The partners may be compoundsthat bind to particular macromolecules of interest and are capable ofactivating or inhibiting the biological macromolecules of interest. Ingeneral, this method involves (1) providing an array of one or moretypes of compounds, as described above, wherein the array of smallmolecules has a density of at least 1000 spots per cm²; (2) contactingthe array with one or more types of biological macromolecules ofinterest; and (3) determining the interaction of specific smallmolecule-biological macromolecule partners.

It will also be appreciated that the arrays of the present invention maybe utilized in a variety of ways to enable detection of interactionsbetween small molecules and biological macromolecules. In oneparticularly preferred embodiment, an array of different types ofchemical compounds attached to the surface is utilized and is contactedby one or a few types of biological macromolecules to determine whichcompounds are capable of interacting with the specific biologicalmacromolecule(s). As one of ordinary skill in the art will realize, ifmore than one type of compound is utilized, it is desirable to utilize amethod for encoding each of the specific compounds so that a compoundhaving a specific interaction can be identified. Specific encodingtechniques have been recently reviewed and these techniques, as well asother equivalent or improved techniques, can be utilized in the presentinvention (see, Czarnik, A. W. Current Opinion in Chemical Biology 1997,1, 60; incorporated herein by reference). Alternatively the arrays ofthe present invention may comprise one type of chemical compound and alibrary of biological macromolecules may be contacted with this array todetermine the ability of this one type of chemical compound to interactwith a variety of biological macromolecules. As will be appreciated byone of ordinary skill in the art, this embodiment requires the abilityto separate regions of the support, utilizing paraffin or other suitablematerials, so that the assays are localized.

As one of ordinary skill in the art will realize, the biologicalmacromolecule of interest may comprise any biomolecule. In preferredembodiments, the biological macromolecule of interest comprises aprotein, and more preferably the array is contacted with a library ofrecombinant proteins of interest. In yet another preferred embodiment,the biological molecules of interest are provided in the form of celllysates such as those of tumor-associated cells. As will be appreciatedby one of ordinary skill in the art, these proteins may comprisepurified proteins, pools of purified proteins, and complex mixtures suchas cell lysates, and fractions thereof, to name a few. Examples ofparticularly preferred biological macromolecules to study include, butare not limited to those involved in signal transduction, dimerization,gene regulation, cell cycle and cell cycle checkpoints, and DNA damagecheckpoints. Furthermore, the ability to construct libraries ofexpressed proteins from any organism or tissue of interest will lead tolarge arrays of recombinant proteins. The compounds of interest may becapable of either inactivating or activating the function of theparticular biomolecule of interest.

Each of the biological macromolecules may be modified to enable thefacile detection of these macromolecules and the immobilized compounds.This may be achieved by tagging the macromolecules with epitopes thatare subsequently recognized, either directly or indirectly, by adifferent receptor (e.g., an antibody) that has been labeled forsubsequent detection (e.g., with radioactive atoms, fluorescentmolecules, colored compounds, or enzymes that enable color formation, orlight production, to name a few). Alternatively, the macromoleculesthemselves may be labeled directly using any one or other of thesemethods or not labeled at all if an appropriate detection method is usedto detect the bound protein (e.g., mass spectrometry, surface plasmonresonance, and optical spectroscopy, to name a few).

In a particularly preferred embodiment, the inventive arrays areutilized to identify compounds for chemical genetic research. Inclassical genetics, either inactivating (e.g., deletion or “knock-out”)or activating (e.g., oncogenic) mutations in DNA sequences are used tostudy the function of the proteins that are encoded by these genes.Chemical genetics instead involves the use of small molecules that alterthe function of proteins to which they bind, thus either inactivating oractivating protein function. This, of course, is the basis of action ofmost currently approved small molecule drugs. The present inventioninvolves the development of “chip-like” technology to enable the rapiddetection of interactions between small molecules and specific proteinsof interest. The examples presented below demonstrate how the methodsand compositions of the present invention can be used to identify newsmall molecule ligands for use in chemical genetic research. One ofordinary skill in the art will realize that the inventive compositionsand methods can be utilized for other purposes that require a highdensity chemical compound format.

As will also be appreciated by one of ordinary skill in the art, arraysof chemical compounds may also be useful in detecting interactionsbetween the compounds and alternate classes of molecules other thanbiological macromolecules. For example, the arrays of the presentinvention may also be useful in the fields of catalysis and materialsresearch to name a few.

These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the claims.

EXAMPLES Example 1 Small Molecule Printing Using Michael Addition

In order to demonstrate the utility of small molecule printing as atechnique identifying small molecule-protein interactions, threeunrelated molecules were chosen for which specific protein receptors areavailable. Compound 1 (FIG. 6, R═OH) is the vitamin biotin, which isrecognized by the bacterial protein streptavidin (Chaiet et al., Arch.Biochem. Biophys. 1964, 106, 1; incorporated herein by reference).Compound 2 (R═OH) is a derivative of the steroid digoxigenin and isrecognized by the mouse monoclonal antibody DI-22 (Sigma). Finally,compound 3 (R═OH) is a synthetic pipecolyl α-ketoamide, which wasdesigned to be recognized by the human immunophilin FKBP12 (Holt et al.,J. Am. Chem. Soc. 1993, 115, 9925; incorporated herein by reference).Each of these compounds was attached to 400-450 μm diameter polystyrenebeads (estimated capacity of 20 nmol per bead) via a 6-aminocaproic acidlinker and either 4-methoxytrityl-protected cysteine (FIG. 6, X═S(Mmt))or alanine (FIG. 6, X═H; negative control). To create reference pointson the slides, beads were also prepared with a thiol-labeled derivativeof the fluorescent dye tetramethylrhodamine (4a). Individual beads wereplaced in 28 separate wells of a 96-well plate and the compounds weredeprotected, cleaved, and subsequently dissolved in 5 μL of DMF. Thereleased compounds were then arrayed robotically onto a series ofmaleimide-derivatized glass slides with a distance of 300 μm between thecenters of adjacent spots. Each slide was printed according to thepattern illustrated in FIG. 7D. Following a 12 hour room temperatureincubation, the slides were washed extensively and probed with differentproteins.

The slide in FIG. 7A was probed with Cy5-conjugated streptavidin,washed, and subsequently scanned using an ArrayWoRx fluorescence slidescanner. The slide was scanned for both tetramethylrhodaminefluorescence (false-colored green) and Cy5 fluorescence (false-coloredred). As anticipated, only the spots containing 1a were visible whenscanned for Cy5 fluorescence, indicating that localization ofstreptavidin on these spots was both specific for biotin and dependenton the thiol functionality (compound 1b, which lacks a thiol, does notattach to the slide). Using a two-step detection method, the slide inFIG. 7B was probed first with DI-22 and then with a Cy5-conjugatedgoat-anti-mouse antibody (which recognizes DI-22). As anticipated, theCy5 fluorescence localized to the 2a-containing spots. Finally, theslide in FIG. 7C was probed using a three-step method: (His)₆-FKBP12followed by mouse-anti-RGS(His)₆ antibody followed by Cy5-conjugatedgoat-anti-mouse antibody. As before, the fluorescence localized to theappropriate spots.

These results clearly illustrate both the high selectivity andremarkable sensitivity of this slide-based assay. To illustrate thehighly parallel nature of small molecule printing, compound 1a wasreleased from a single 400-450 μm diameter polystyrene bead and thereleased compound was dissolved in 10 μL of DMF. We repeated thisprocedure for compounds 2a and 3a. Using the microarraying robot, thesethree compounds were repetitively spotted in an alternating pattern on asingle maleimide-derivatized slide, using the same spatial density as inFIG. 7. Each compound was spotted 3600 times, using less than half ofthe compound from each bead (˜1 nL per spot) and yielding 10,800distinct spots. The slide was then probed in a single step with asolution containing FITC-conjugated streptavidin, Cy3-conjugated DI-22,and Cy5-conjugated FKBP12. Following a brief washing step, the slide wasscanned for FITC fluorescence (false colored blue), Cy3 fluorescence(false-colored green), and Cy5 fluorescence (false-colored red). Asshown in FIG. 8, the three differently labeled proteins localized to thespots containing their cognate ligands.

Experimental details for the above described example can be found inbelow. One of ordinary skill in the art will realize that the inventivecompositions and methods are not limited to the examples describedabove; rather the present invention is intended to include allequivalents thereof.

Example 2 Small Molecule Printing Using Silylation Reaction

Standard glass slides were activated for selective reaction withalcohols (FIG. 9). Microscopic slides were first treated with aH₂SO₄/H₂O₂ solution (“piranha”) for 16 hours at room temperature. Afterextensive washing with water, the slides were treated with thionylchloride and a catalytic amount of DMF in THF for 4 hours at roomtemperature. Surface characterization by x-ray photoelectronspectroscopy (XPS) confirmed the presence of chlorine on the slide(Strother et al., J. Am. Chem. Soc., 2000, 122, 1205-1209; incorporatedherein by reference). To test the ability of these chlorinated slides tocapture alcohols released from synthesis beads, we initially used threealcohol-containing small molecules and a bead linker reagent developedfor chemical genetic applications of diversity-oriented synthesis.

Primary alcohol derivatives of a synthetic α-ketoamide (Holt et al., J.Am. Chem. Soc. 1993, 115, 9925-9938; incorporated herein by reference),digoxigenin, and biotin were attached to silicon linker-modified beads(FIG. 10). These beads are high capacity 500-560 polystyrene beadsequipped with an all hydrocarbon and silicon linker for the temporaryattachment and eventual fluoride-mediated release of synthetic,alcohol-containing compounds. The three primary alcohol derivatives haveknown protein partners, namely FKBP12 (Harding et al., Nature, 1989,341, 758-760; Siekierkea et al., Nature, 1989, 341, 755-757; each ofwhich is incorporated herein by reference), the DI-22 antibody (Sigma),and streptavidin (Chaiet et al., Arch. Biochem. Biophys., 1964, 106,1-5; incorporated herein by reference), respectively. AfterHF-pyridine-mediated release from the beads and subsequent solventremoval, the compounds were dissolved in 5 μL of DMF in individual wellsof 96-well plates to give ˜5 mM solutions. A microarrayer was used tospot the compounds (in triplicate) 400 μm apart (average spot diameterof 300 μm) onto the thionyl chloride-activated slides (FIG. 10 b-e) andthe slides were then washed extensively with DMF, THF, isopropanol, andan aqueous buffer. As shown, when binding was detected separately (FIG.10 b-d) or simultaneously (FIG. 10 e), the recognition of the proteinfor its ligand was efficient and selective. When the same compounds wereprinted onto control slides (i.e., not activated with thionyl chloride)no protein-ligand interactions were detected.

Small molecules resulting from diversity-oriented syntheses can containa wide array of functional groups, including secondary and phenolichydroxyls. To test the ability of such functionalities to react with thethionyl chloride activated slides, the synthetic α-ketoamide derivativesshown in FIG. 11 were synthesized. An array was then printed (inquadruplicate) containing the primary, secondary, phenolic, and methylether derivatives at ˜5 mM, and probed with Cy5-FKBP. As shown in FIG.11, the reaction of the primary alcohol is favored, and this bias holdseven when the secondary, phenolic, and methyl ether derivatives arearrayed at a concentration ten times greater than the primary.

As a demonstration of the compatibility of this alcohol arrayingtechnique with split-pool synthesis, a collection of 78 small moleculesderived from such synthesis having the general structure, shown in FIG.12 a was printed onto glass slides (Tan et al., J. Am. Chem. Soc. 1998,120, 8565-8566; incorporated herein by reference). To this collectionwere added two members that had been acylated with the syntheticα-ketoamide derivative or biotin (FIG. 12 b). These ‘tagged’ memberswere then released from their beads, dissolved in 5 μL of DMF, andplaced in known wells of a 96-well plate. After placing the 80 compoundsinto discrete wells, the entire plate was arrayed onto thionylchloride/DMF activated slides, which were then probed withfluorescently-labeled proteins, Cy5-FKBP12 and FITC-streptavidin. Theresults (FIG. 12 c) show that two spots in the array fluoresce in theCy5 channel (false-colored red), and another fluoresces in the FITCchannel (false-colored green). The positional encoding confirms theresult that the compound acylated with the α-ketoamide was spotted inB8, and the compound acylated with biotin was spotted in F2. The spotvisible in E3 is an apparent serendipitous and reproducible ‘hit’, andawaits further analysis. Thus, this experiment demonstrates the processof split-pool synthesis, release from the solid support, arraying ontoglass slides, and detection/visualization of protein-small moleculebinding events.

Example 3 Fabrication of Custom Slide Reaction Vessels

In an effort to minimize reagent volume during the chemical treatment ofglass microscope slides, we designed and fabricated custom slide-sizedreaction vessels that enable the uniform application of ˜1.4 mL solutionto one face of a 2.5 cm×7.5 cm slide. First, a master template mold wascut from a block of Delhran plastic according to the blueprint shown inFIG. 13. The slide-sized reaction vessels were prepared by castingdegassed polydimethysiloxane (PDMS, Sylgard Kit 184, Dow coming,Midland, Mich.) prepolymer around the master template in a polystyreneOmniTray (Nalge Nunc International, Naperville, Ill.). After curing forfour hours at 65° C., the polymer was peeled away from the master togive the finished product (FIG. 14).

To use the vessels, slides were placed face-down as illustrated belowand reagent was injected under the slides with a P1000 Pipetman (FIG.15).

Example 4 Chemical Derivatization of Glass Microscope Slides

Plain glass slides (VWR Scientific Products, USA) were cleaned in a“piranha” solution (70:30 v/v mixture of concentrated H₂SO₄ and 30%H₂O₂) for 12 hours at room temperature. (Caution: “piranha” solutionreacts violently with several organic materials and should be handledwith extreme care (Pintochovski et al., Electrochem. Soc. 1979, 126,1428; Dobbs et al., Chem. Eng. News 1990, 68(17), 2; Wnuk, Chem. Eng.News 1990, 68(26), 2; Erickson, Chem. Eng. News 1990, 68(33), 2; each ofwhich is incorporated herein by reference)). After thorough rinsing withdistilled water, the slides were treated with a 3% solution of3-aminopropyltriethoxysilane (United Chemical Technologies, Bristol,Pa.) in 95% ethanol for 1 hour. (Before treating the slides, the 3%silane solution was stirred for at least 10 minutes to allow forhydrolysis and silanol formation). The slides were then briefly dippedin 100% ethanol and centrifuged to remove excess silanol. The adsorbedsilane layer was cured at 115° C. for one hour. After cooling to roomtemperature, the slides were washed several times in 95% ethanol toremove uncoupled reagent.

A simple, semi-quantitative method was used to verify the presence ofamino groups on the slide surface (Licitra et al., Proc. Natl. Acad.Sci. USA 1996, 93, 12817-12821; incorporated herein by reference). Oneglass slide from each batch of amino-functionalized slides was washedbriefly with 5 mL of 50 mM sodium bicarbonate, pH 8.5. The slide wasthen dipped in 5 mL of 50 mM sodium bicarbonate, pH 8.5 containing 0.1mM sulfo-succinimidyl-4-O-(4,4′-dimethoxytrityl)-butyrate (s-SDTB;Pierce, Rockford, Ill.) and shaken vigorously for 30 minutes. (Thes-SDTB solution was prepared by dissolving 3.03 mg of s-SDTB in 1 mL ofDMF and diluting to 50 mL with 50 mM sodium bicarbonate, pH 8.5). Aftera 30 minute incubation, the slide was washed three times with 20 mL ofdistilled water and subsequently treated with 5 mL of 30% perchloricacid. The development of an orange-colored solution indicated that theslide had been successfully derivatized with amines; no color change wasseen for untreated glass slides. Quantitation of the4,4′-dimethoxytrityl cation ε_(498nm)=70,000 M⁻¹cm⁻¹) released by theacid treatment indicated an approximate density of two amino groups pernm².

The resulting amino-functionalized slides were transferred to customslide-sized polydimethylsiloxane (PDMS) reaction vessels (as describedin Example 3). One face of each slide was treated with 20 mMN-succinimidyl 3-maleimido propionate (Aldrich Chemical Co., Milwaukee,Wis.) in 50 mM sodium bicarbonate buffer, pH 8.5, for three hours. (Thissolution was prepared by dissolving the N-succinimidyl 3-maleimidopropionate in DMF and then diluting 10-fold with buffer). Afterincubation, the plates were washed several times with distilled water,dried by centrifugation, and stored at room temperature under vacuumuntil further use.

Example 5 Attachment of Small Molecules to Polystyrene Beads Materials

Fmoc-εAhx-OH and PyBOP® were from Novabiochem (San Diego, Calif.).

Biotin and diisopropylethylamine (DIPEA) were from Aldrich Chemical Co.(Milwaukee, Wis.). 3-Amino-3-deoxydigoxigenin hemisuccinamide,succinimidyl ester and 5(6)-TAMRA, SE were from Molecular Probes(Eugene, Oreg.). Wash solvents were obtained from Mallinckrodt or E.Merck and used as received. Anhydrous dimethylformamide (DMF) wasobtained from Aldrich Chemical Co. in SureSeal™ bottles.

The “FKBP Ligand” is shown below and was synthesized as published(Keenan et al., Bioorg. Med. Chem. 1998, 6, 1309; Amara et al., Proc.Natl. Acad. Sci. USA 1997, 94, 10618-10623; each of which isincorporated herein by reference).

Polystyrene synthesis beads were obtained by custom synthesis from RappPolymere (Tübingen, Germany). They ranged from 400 μm to 450 μm indiameter, had an estimated capacity of about 0.4 mmol/g (17 nmol/bead),and came functionalized as indicated below.

Solid Phase Reactions.

Solid phase reactions were performed in either 2 mL frittedpolypropylene Biospin® chromatography columns (Pharmacia Biotech,Uppsala, Sweden) or 10 mL flitted polypropylene PD-10 columns (PharmaciaBiotech). Resin samples were washed on a Val-Man® Laboratory VacuumManifold (Promega, Madison, Wis.) using the following procedure: 3×DMF,3×THF, 3×DMF, 3×THF, 3×DMF, 3×THF, 3×DMF, 6×CH₂Cl₂, 3×THF.

Polystyrene Beads with Attached Linker (5c, 5d).

Either Polystyrene A Trt-Cyc(Mmt) Fmoc or Polystyrene A Trt-Ala Fmoc(400 mg, 0.4 mmol/g, 0.16 mmol) was placed in a 10 mL column and swollenwith 6 mL DMF for 2 min. The column was drained and the Fmoc groupremoved by two 15 min treatments with 6 mL of 20% piperidine in DMF. Theresin was washed (as described above), dried under vacuum, and swollenwith 6 mL anhydrous DMF for 2 min. The column was drained and the resinswollen with 6 mL distilled CH₂Cl₂ for another 2 min. The column wasdrained and a mixture containing anhydrous DMF (5.2 mL), Fmoc-εAhx-OH(283 mg, 0.80 mmol, 5 eq), PyBOP® (416 mg, 0.80 mmol, 5 eq), and DIPEA(279 μL, 160 mmol, 10 eq) was added. After 12 h, the resin was washedand found to be negative to Kaiser ninhydrin test. The Fmoc group wasthen removed (as above) and the resin washed to give 5c and 5d.

Polystyrene Beads with Attached Linker and Biotin (1c, 1d).

Either resin 5c or resin 5d (100 mg, 0.040 mmol, 1 eq) was placed in a 2mL column and swollen with 1.5 mL anhydrous DMF for 2 min. The columnwas drained and the resin swollen with 1.5 mL distilled CH₂Cl₂ foranother 2 min. The column was drained and a mixture containing anhydrousDMF (1.3 mL), biotin (39.1 mg. 0.16 mmol, 4 eq), PyBOP® (83.3 mg, 0.16mmol, 4 eq), and DIPEA (55.7 μL, 0.32 mmol, 8 eq) was added. After 12 h,the resin was washed and subsequently found to be negative to Kaiserninhydrin test.

Polystyrene Beads with Attached Linker and Digoxigenin Derivative (2c,2d).

Either resin 5c or resin 5d (10 mg, 0.004 mmol, 1 eq) was placed in a 2mL column and swollen with 1.5 mL anhydrous DMF for 2 min. The columnwas drained and the resin swollen with 1.5 mL distilled CH₂Cl₂ foranother 2 min. The column was drained and a mixture containing anhydrousDMF (1.0 mL), 3-amino-3-deoxydigoxigenin hemisuccinamide, succinimidylester (5.0 mg, 0.0085 mmol, 2.1 eq), and DIPEA (20 μL, 0.115 mmol, 29eq) was added. After 12 h, the resin was washed and treated for anadditional 12 h with a fresh preparation of the mixture described above.The resin was washed again and subsequently found to be negative toKaiser ninhydrin test.

Polystyrene Beads with Attached Linker and FKBP Ligand (3c, 3d).

Either resin 5c or resin 5d (100 mg, 0.04 mmol, 1 eq) was placed in a 2mL column and swollen with 1.5 mL anhydrous DMF for 2 min. The columnwas drained and the resin swollen with 1.5 mL distilled CH₂Cl₂ foranother 2 min. The column was drained and a mixture containing anhydrousDMF (1.3 mL), FKBP ligand (67.5 mg, 0.116 mmol, 2.9 eq), PyBOP® (83.3mg, 0.16 mmol, 4 eq), and DIPEA (55.7 μL, 0.32 mmol, 8 eq) was added.After 12 h, the resin was washed and subsequently found to be negativeto Kaiser ninhydrin test.

Polystyrene Beads with Attached Linker and TetramethylrhodamineDerivative (4c).

Either resin 5c or resin 5d (40 mg. 0.016 mmol, 1 eq) was placed in a 2mL column and swollen with 1.5 mL anhydrous DMF for 2 min. The columnwas drained and the resin swollen with 1.5 mL distilled CH₂Cl₂ foranother 2 min. The column was drained and a mixture containing anhydrousDMF (1.0 mL), 5(6)-TAMRA, SE (25 mg, 0.047 mmol, 3.0 eq), and DIPEA (20μL, 0.115 mmol, 7.2 eq) was added. After 12 h, the resin was washed andtreated for an additional 12 h with a fresh preparation of the mixturedescribed above. The resin was washed again to yield resin 4c.

Mass Spectrometry.

As confirmation of this standard coupling chemistry, about 10 beads eachof 1c, 1d, 2c, 2d, 3c and 3d were exposed to 100 μL of trifluoroaceticacid/triisopropylsilane/chloroform (2:1:17) for 2 h at room temperature.The deprotection/cleavage solution was then removed in vacuo and theliberated compounds dissolved in 20 μL DMF. FAB⁺ MS gave molecularweights that exactly matched those predicted for compounds 1a, 1b, 2a,2b, 3a and 3b, respectively.

Example 6 Small Molecule Printing

Deprotection and Release of Small Molecules.

Individual beads (1c, 1d, 2c, 2d, 3c, 3d, 4c) were placed in separatewells of a polypropylene V-bottom 96-well plate (Costar, Corning, N.Y.)using an 18-gauge needle and a low power dissecting microscope. To eachwell was added 20 μL of trifluoroacetic acid/triethylsilane/chloroform(2:1:17) and the wells were immediately sealed with polyethylene stripcaps (Nalge Nunc International, Naperville, Ill.). After 2 h at roomtemperature, the caps were discarded and the cleavage solution removedin vacuo. The released compounds were then dissolved in 5-10 μL of DMFand printed onto maleimide-derivatized glass slides.

Robotic Arraying of Small Molecules.

Small molecules were printed using a microarraying robot (FIGS. 16, 17,and 18), constructed in this laboratory by Dr. James S. Hardwick andJeffrey K. Tong according to directions provided by Dr. Patrick O. Brown(http://cmgm.stanford.edu/pbrown/mguide/index.html).

The robot was instructed to pick up a small amount of solution (˜250 nL)from consecutive wells of a 96-well plate and repetitively deliverapproximately 1 nL to defined locations on a series ofmaleimide-derivatized glass microscope slides. The pin used to deliverthe compounds was washed with double distilled water for 8 s and driedunder a stream of air for 8 s before loading each sample (6 s).Following printing, the slides were incubated at room temperature for 12h and then immersed in a solution of 2-mercaptoethanol/DMF (1:99) toblock remaining maleimide functionalities. The slides were subsequentlywashed for 1 h each with DMF, THF, and iPrOH, followed by a 1 h aqueouswash with MBST (50 mM MES, 100 mM NaCl, 0.1% Tween® 20, pH 6.0). Slideswere rinsed with double-distilled water, dried by centrifugation, andeither used immediately or stored at room temperature for several dayswithout any observed deterioration.

Example 7 Detection of Protein-Small Molecule Interactions

Materials.

Cy5-streptavidin, Cy5-goat-anti-mouse IgG, and FITC-streptavidin werefrom Kirkegaard & Perry Laboratories (Gaithersburg, Md.).Mouse-anti-digoxin IgG (DI-22) was from Sigma-Aldrich Co. (St. Louis,Mo.). Mouse-anti-(His)₆IgG (RGSHis antibody) was from Qiagen (Hilden,Germany).

Production of (His)₆-FKBP12.

Construction of T5 Expression Plasmid.

A 355-bp PCR product containing the coding sequence for human FKBP12 wasobtained using primers FKBP-1S (ACGTACGTGGATCCATGGGAGTGCAGGTGGAAACCA)and FKBP-1N (ACGTACGTGTCGACTTATTCCAGTTTTAGAAGCTCCACATCGA) on templatepJG-FKBP12 (Licitra et al., Proc. Natl. Acad. Sci. USA 1996, 93,12817-12821; incorporated herein by reference). The 333-bp Bam HI-Sal Ifragment of this product was then ligated with the 3434-bp Bam HI-Sal Ifragment of pQE-30 (Qiagen) to yield the T5 expression plasmidpQE-30-FKBP12 (3757 bp).

Production and Purification of (His)₆ FKBP 12.

The host strain for protein production was M15[pREP4] (Qiagen). Cellsfrom a single colony were grown in 500 mL of LB medium supplemented with100 μg/mL sodium ampicillin and 25 μg/mL kanamycin at 37° C. up to anOD₆₀₀ of 0.8. The culture was cooled to room temperature and isopropyl1-thio-β-D-galactopyranoside (IPTG) was added to a final concentrationof 1 mM. After 16 h induction at room temperature, the cells wereharvested and resuspended in 20 mL of PBS (10 mM phosphate, 160 mM NaCl,pH 7.5) supplemented with 100 μM phenylmethanesulfonyl fluoride (PMSF).Following cell lysis by passage through a French press, insolublematerial was removed by centrifugation (28000 g, 20 min, 4° C.) and thesupernatant loaded onto a column packed with 5 mL of Ni-NTA agarose(Qiagen) that had been preequilibrated with PBS. The column wasthoroughly washed with PBS containing 10 mM imidazole, and bound proteinwas subsequently eluted with PBS containing 250 mM imidazole. The samplewas dialyzed extensively against PBS and stored at 4° C.

Labeling of Proteins with Fluorophores.

Cy3-labeled DI-22 was prepared from DI-22 mouse ascites fluid(Sigma-Aldrich Co.) using FluoroLink™ Cy3™ bisfunctional reactive dye(Amersham Pharmacia Biotech, Piscataway, N.J.) according to therecommended protocol. Similarly, Cy5-labeled (His)₆-FKBP12 was preparedfrom purified (His)₆-FKBP12 using FluoroLink™ Cy5™ monofunctionalreactive dye (Amersham Pharmacia Biotech) according to the recommendedprotocol.

Probing Slides with Proteins.

Reagents were applied to the printed face of the slides using PDMS slidereaction chambers. Rinsing and washing steps were performed with theslides face up in the lids of pipet tip boxes.

In each experiment, the slides were blocked for 1 h with MBSTsupplemented with 3% bovine serum albumin (BSA). Following each step inthe procedure, the slides were rinsed briefly with MBST before applyingthe next solution. With the exception of the blocking step, the slideswere exposed to protein solutions for 30 min at room temperature. Thesesolutions were prepared by diluting stock solutions of the appropriateprotein(s) with MBST supplemented with 1% BSA. After the finalincubation, the slides were rinsed once with MBST and then gentlyagitated with 4 changes of MBST over the course of 12 min. The slideswere dried by centrifugation and stored in the dark at room temperature.

The protein concentrations used in the preparation of FIGS. 7 and 8 wereas follows:

-   FIG. 7A: 1 μg/mL Cy5-streptavidin-   FIG. 7B: 2 μg/mL DI-22 (IgG1)    -   1 μg/mL Cy5-goat-anti-mouse IgG-   FIG. 7C: 40 μg/mL (His)₆-FKBP12    -   2 μg/mL mouse RGSHis IgG    -   1 μg/mL Cy5-goat-anti-mouse IgG-   FIG. 8: 2 μg/mL FITC-streptavidin,    -   +0.2 μg/mL Cy3-DI-22 (IgG1)    -   +4 μg/mL Cy5-(His)₆-FKBP12

Scanning Slides for Fluorescence.

Slides were scanned using an ArrayWoRx™ slide scanner (AppliedPrecision,Issaquah, Wash.). Slides were scanned at a resolution of 5 μm per pixel.Double filters were employed for both the incident and emitted light.For the images in FIG. 7, tetramethylrhodamine fluorescence was observedusing a Cy3/Cy3 excitation/emission filter set (1 s exposure) and Cy5fluorescence was observed using a Cy5/Cy5 excitation/emission filter set(2 s exposure). For the image in FIG. 8, fluorescein fluorescence wasobserved using a FITC/FITC excitation/emission filter set (10 sexposure), Cy3 fluorescence was observed using a Cy3/Cy3excitation/emission filter set (2 s exposure), and Cy5 fluorescence wasobserved using a Cy5/Cy5 excitation/emission filter set (5 s exposure).The full slide image (top) was stitched with 4-fold pixel reduction andthe magnified image (bottom) was stitched with 2-fold pixel reduction.

Example 8 Covalent Attachment and Screening of Alcohol-Containing SmallMolecules on Glass Slides

General Procedures for Synthetic Transformations:

Methylene chloride, diisopropylethylamine and dimethylformamide weredistilled under nitrogen from calcium hydride. Tetrahydrofuran (HPLCgrade, Fisher, solvent keg) was dried by passing the solvent through twocolumns of activated alumina (A-2) (Panghom et al., Organometallics1996, 15, 1518; incorporated herein by reference). All other reagentswere obtained from commercial suppliers. Solution phase reactions werecarried out in 2 dram vials with Teflon screw caps. Reactions weremonitored by thin layer chromatography using 0.25 mm silica gel 60 F₂₅₄plates from EM Science and visualized with ceric ammonium molybdate(CAM) stain. All compounds were purified using 230-400 mesh silica gel60 from EM Science. Biotinol was prepared as previously described (Islamet al., J. Med. Chem. 1994, 37, 293-304; incorporated herein byreference). The digoxigenin derivative as its N-hydroxysuccinimide esterwas obtained from Molecular Probes Inc. The FKBP ligand (AP1497, anacid) was obtained from Dr. Kazunori Koide of Harvard University andfrom Ariad Pharmaceuticals (Keenan et al., Bioorg. Med. Chem. Lett.1998, 6, 1309; incorporated herein by reference).

The solid support, 500-560 μm polystyrene 1% divinylbenzene (RappPolymere) was derivatized with a 3-(p-anisolyldiisopropylsilyl)-propyllinker (Woolard et al., J. Org. Chem. 1997, 62, 6102; incorporatedherein by reference). The library members were obtained from Dr. KoujiHattori (Harvard). The secondary alcohol of this scaffold wasderivatized following the method of Tan et al. (J. Am. Chem. Soc. 1999,121, 9073-9087; incorporated herein by reference). Solid phase loadingreactions were run under an inert atmosphere in 2.0 mL polypropyleneBio-Spin® chromatography columns (Bio-Rad Laboratories, Hercules,Calif.; 732-6008) bearing a 3-way nylon stopcock (Bio-Rad; 732-8107) andmixed by 360° rotation on a Bamstead-Thermolyne Labquake Shaker™ (VWR56264-306).

Representative Procedure for the Synthesis of the FKBP Ligands.

To the above mentioned acid (17 mg, 0.029 mmol), in a solution of DMF(0.30 mL) was added the respective amine or amine hydrochloride (0.038mmol), PyBOP (24.4 mg, 0.047 mmol), and i-Pr₂NEt (0.015 mL, 0.088 mmol,amines; 0.020 mL, 0.12 mmol, amine hydrochlorides). The solution wasstirred at ambient temperature for 15 h, dissolved in a dilute brinesolution and was extracted with EtOAc (3 times). The organic layers werecombined, washed with a 1/1 water/saturated brine solution, dried overNa₂SO₄, filtered, concentrated, and chromatographed on silica gel (0 to10% in CHCl₃) to give a colorless film.

primary OH (1) (reaction with ethanolamine); ¹H NMR (500 MHz, CDCl₃) δ7.28 (m, 1H), 7.18 (m, 1H NH), 6.98-6.66 (m, 6 H), 5.75 (dd, J=7.8, 5.4Hz, 1H), 5.29 (d, J=4.9 Hz, 1H), 4.51 (m, 2 H), 3.85 (s, 3 H), 384 (s, 3H), 3.72 (m, 2 H), 3.51 (m, 2 H), 3.35 (b d, J=13.2 Hz 1H), 3.16 (td,J=12.3, 2.7 Hz, 1H), 2.56 (m, 2 H), 2.36 (b d, J=13.7 Hz, 1H), 2.23 (m,1H), 2.05 (m, 1H), 1.77-1.62 (m, 6H), 1.48 (m, 1H), 1.34 (m, 1H), 1.21(s, 3H), 1.19 (s. 3 H), 0.87 (t, J=7.6 Hz, 3 H); HRMS (TOF-ES⁺) cal. forC₃₄H₄₇N₂O₉(M+H)⁺, 627.3282, obs. 627.3306.

primary OMe (reaction with 2-methoxyethylamine); ¹H NMR (500 MHz, CDCl₃)δ 7.30 (m, 1 H), 7.00-6.67 (m, 7 H), 5.76 (m, 1H), 5.32 (d, J=4.9 Hz,1H), 4.51 (m, 2 H), 3.86 (s, 3 H), 3.85 (s, 3 H), 3.55 (m, 2 H), 3.49(m, 2H), 3.37 (d, J=13.0 Hz, 1H), 3.35 (s, 3H), 3.16 (td, J=13.2, 2.9Hz, 1H), 2.57 (m, 2 H), 2.36 (b d, J=13.7 Hz 1H), 2.24 (m, 1H), 2.06 (m,1H), 1.79-1.58 (m, 6 H), 1.51-1.30 (m, 2 H), 1.23 (s, 3 H), 1.21 (s,3H), 0.89 (t, J=7.6 Hz, 3 H); HRMS (TOF-ES⁺) calc. forC₃₅H₄₈N₂O₉Na(M+Na)⁺, 663.3258, obs. 663.3229.

secondary OH (reaction with trans-4-amino-cyclohexanol hydrochloride);¹H NMR (500 MHz, CDCl₃) δ 7.29 (m, 1H), 6.99-666 (m, 6H), 6.41 (d, J=8.3Hz, 1H, NH), 5.76 (M, 1H), 5.30 (d, J=5.4 Hz, 1H), 4.46 (m, 2H), 3.85(s, 3 H), 3.84 (s, 3H), 3.61 (m, 1H), 3.36 (b d, J=12.2 Hz, 1H), 3.20TD, J=13.2, 2.9 Hz, 1H), 2.56 (m, 2H), 2.36 (b d, J=13.7 Hz, 1H), 2.24(m, 1H), 2.00 (m, 4H), 1.78-1.61 (m, 6H), 1.50-1.23 (m, 4H), 1.21 (s,3H), 1.20 (s, 3H), 0.88 (t, J=7.3 Hz, 3 H); HRMS (TOF-ES⁺) calc. forC₃₈H₅₃N₂O₉(M+H)⁺, 681.3751, obs. 681.3778.

phenolic OH (reaction with tyramine hydrochloride); ¹H NMR (500 MHz,CDCl₃) δ 7.31 (m, 1 H), 7.01-66 (m, 10 H), 6.51 (m, 1H, NH), 5.81 (m,1H), 5.33 (b d, J=5.1 Hz, 1H), 4.60 (m, 2H), 3.86 (s, 6 H), 3.55 (m, 2H), 3.40 (b d, J=13.0 Hz 1H), 3.27 (td, J=13.2, 2.9 Hz, 1H), 2.72 (t,J=6.4 Hz, 2 H), 2.56 (m, 2 H), 2.40 (b d, J=13.2 Hz, 1H), 2.24 (m, 1 H),2.06 (m, 1H), 1.87-1.64 (m, 6 H), 1.54 (m, 1H), 1.40 (m, 1H), 1.24 (s, 3H), 1.21 (s, 3 H), 0.88 (t, J=7.5 Hz, 3H); HRMS (TOF-ES⁺) calc. forC₄₀H₅₀N₂O₉Na(M+Na)⁺, 725.3414, obs. 725.3384.

Procedure for the Digoxigenin Derivative.

To a solution of the NHS ester of the digoxigenin derivative (5.0 mg,0.0085 mmol) in DMF (0.3 mL) was added ethanolamine (0.0008 mL, 0.013mmol) and 4-methylmorpholine (0.0011 mL, 0.010 mmol). The reaction wasstirred at ambient temperature for three days, concentrated under highvacuum at room temperature, and chromatographed on silica gel (0 to 20%MeOH in CHCl₃); ¹H NMR (400 MHz, 5/1 CDCl₃/CD₃OD) δ 5.84 (s, 1H), 4.81(AB d, 2 H), 4.00 (b s, 1H) 3.55 (m, 2H) 3.24 (m, 3H), 2.39 (m, 4 H),2.04 (m, 1H), 1.81 (m, 4 H), 1.70-1.38 (m, 9H), 1.16 (m, 6 H), 0.88 (s,3H), 0.67 (s, 3H).

General Procedure for Loading Alcohols Via a Silicon Ether ontoPolystyrene Beads.

After drying under vacuum for 8 h, the large polystyrene beads bearing a3-(p-anisolyldiisopropylsilyl)-propyl linker (13.3 mg, 0.008 mmol, ca.0.6 mmol silane/g resin) were added to a Bio-Rad tube, which was cappedwith a septum and a plastic stopcock and flushed with an inert gas. Thetube was then charged via syringe with a 2.5% (v/v) solution of TMS-Clin CH₂Cl₂ The beads were suspended for 15 min, and filtered with inertgas pressure. The beads were washed with CH₂Cl₂ (0.5 mL, 3 times, 2min/rinse) and then suspended in a 3% (v/v) solution of triflic acid inCH₂Cl₂ (0.142 mL, 0.049 mmol) for 15 min during which time the tube wasshaken periodically. The beads turn a dark brown color. The beads weresuspended and rinsed with CH₂Cl₂ (0.5 mL, 3 times, 2 rain/rinse) underan inert gas, and left suspended in the fourth volume of CH₂Cl₂ Freshlydistilled 2,6-lutidene (0.007 mL, 0.064 mmol) was added (the brown colordisappears) and the azeotropically dried (from benzene) alcohol (0.020mmol) was added as a solution in CH₂Cl₂ via a canula transfer (forα-ketoamide and digoxigenin, 3 volumes, 0.3 mL/transfer) or introducedas a neat solid (e.g., biotinol, when the alcohol is not soluble inCH₂Cl₂). The tube was capped and tumbled at ambient temperature for 2-4h. The beads were then filtered, suspended, and rinsed, for α-ketoamide,with CH₂Cl₂ (10 times, 5 min/rinse) and dried under high vacuum; fordigoxigenin and biotin, the beads were rinsed likewise with DMF toremove non-covalently attached ligand.

Activation of Slides for Microarraying.

Slides were activated for covalent attachment of alcohols as follows.Standard microscope slides (VWR) were immersed in 70/30 (v/v) H₂SO₄/30%H₂O₂ (piranha) for 16 h at ambient temperature. After removal from thepiranha bath, the slides were washed extensively in ddH₂O, and then keptunder water until use. To convert to the silyl chloride, the slides werefirst removed from the water and dried by centrifugation. At this point,the slides were immersed in a solution of THF containing 1% SOCl₂ and0.1% DMF. The slides were incubated in this solution for 4 h at ambienttemperature. The slides were then removed from the chlorinationsolution, washed briefly with THF, and placed on the microarrayer.

Release of Alcohols from their Solid Supports.

To liberate alcohols from the polystyrene beads, single beads weretreated with 10 μL of 90/5/5 (v/v) THF/HF.pyridine/pyridine at ambienttemperature for 1 h. 10 μL of TMSOMe was then added, and allowed tostand at ambient temperature for an additional 0.5 h. The solvent wasthen removed in vacuo, and the liberated compound from a single bead wasdissolved in 5 μL of DMF. These solutions were then robotically arrayedonto activated glass slides.

We confirmed the coupling of the α-ketoamide and biotin to the secondaryalcohol of the library by LC/LRMS (TOF-ES⁺) analysis of materialreleased from a single bead of each type. The observed ions (M+H)⁺ of1064 and 725 matched the theoretical masses expected for C₅₉H₇₅N₄O₁₄(α-ketoamide) and C₃₇H₅₀N₅O₈S (biotin), respectively.

Robotic Printing.

Compounds were arrayed onto glass slides using a DNA microarrayerconstructed by Dr. James Hardwick and Jeff Tong following instructionson the web site of Professor Patrick Brown (Standard University;http://cmgm.stanford.edu/pbrown/mguide/index.html; incorporated hereinby reference). The microarrayer typically picks up 250 nL from the96-well plate and delivers 1 nL drops onto the slides. These spots wereplaced 400 μm apart on the slides.

Detection of Protein/Ligand Interactions.

After arraying, the slides were allowed to incubate at ambienttemperature for 12 h. The slides were then washed for 2 h with DMF, and1 h each with THF, isopropanol, and MBST (50 mM MES, 100 mM NaCl, 0.1%Tween-20, pH=6.0). The slides were then blocked for 1 h by incubationwith MBST containing 3% BSA. After a brief rinse with MBST, thefluorescently labeled protein was then added at a concentration of 1μg/mL in MBST supplemented with 1% BSA. The labeled proteins werecreated as described (Tan et al., J. Am. Chem. Soc. 1999, 121,9073-9087; MacBeath et al., J. Am. Chem. Soc. 1999, 121, 7967-7968; eachof which is incorporated herein by reference). The slide was incubatedwith the labeled protein for 0.5 h at ambient temperature. At thispoint, the slide was washed (10 times 1 mL with MBST, then briefly withH₂O) and dried by centrifugation. The slide was then scanned using anArrayWoRx slide scanner (AppliedPrecision, Issaquah, WA) at a resolutionof 5 μm per pixel. The following filter sets were employed: Cy5/Cy5excitation/emission filter set (2 s exposure); Cy3/Cy3excitation/emission filter set (1 s exposure); FITC/FITC excitationemission filter set (10 s exposure).

Other Embodiments

Those of ordinary skill in the art will readily appreciate that theforegoing represents merely certain preferred embodiments of theinvention. Various changes and modifications to the procedures andcompositions described above can be made without departing from thespirit or scope of the present invention, as set forth in the followingclaims.

What is claimed is:
 1. An array comprising a plurality of more than onetype of chemical compound attached to a solid support through a linkerof the formula:

wherein the density of the array of chemical compounds is at least 1000spots per cm².
 2. An array comprising a plurality of more than one typeof chemical compound attached to a solid support, wherein the density ofthe array of chemical compounds is at least 1000 spots per cm², andwherein the chemical compounds are attached to the solid support asshown below:

wherein: X is O, S, or NH of the attached chemical compound R; and n is0, 1, 2, 3, 4, or
 5. 3. The array of claim 1, wherein the array ofchemical compounds is an array of small molecules.
 4. The array of claim1, wherein the array of chemical compounds is an array of non-oligomericchemical compounds.
 5. The array of claim 1, wherein the molecularweight of the chemical compounds is less than 1500 g/mol.
 6. The arrayof claim 1, wherein the density of the array of the chemical compoundsis at least 5000 spots per cm².
 7. The array of claim 1, wherein thesolid support comprises a substantially flat surface.
 8. The array ofclaim 1, wherein the solid support is glass.
 9. The array of claim 1,wherein the solid support is derivatized glass.
 10. The array of claim1, wherein the solid support is silylated glass.
 11. The array of claim1 wherein the solid support is γ-aminopropylsilylated glass.
 12. Thearray of claim 1, wherein the solid support is a polymer.
 13. The arrayof claim 1, wherein the solid support is metal.
 14. The array of claim1, wherein the solid support is a metal-coated surface.
 15. The array ofclaim 1, wherein the attachment of the chemical compound to the solidsupport is characterized in that the attachment is robust enough so thatthe chemical compounds are (1) not inadvertently cleaved duringsubsequent manipulation steps and (2) inert so that the functionalitiesemployed do not interfere with subsequent manipulation steps.
 16. Thearray of claim 1, wherein each of the chemical compounds becomesattached to the solid support through a reaction with an isothiocyanatemoiety.
 17. A solid support comprising a solid support derivatized withisothiocyanate moieties.
 18. The solid support of claim 17 comprisingthe structure:

wherein n is 0, 1, 2, 3, 4, or
 5. 19. A method for forming the array ofclaim 1, the method comprising: providing a solid support, wherein thesolid support is derivatized with isothiocyanate moieties capable ofinteracting with more than one type of chemical compound to form acovalent linkage; providing one or more solutions of the more than onetype of chemical compound to be attached to the solid support; anddelivering the one or more solutions of the more than one type ofchemical compound to the solid support, wherein each of the chemicalcompound is attached to the solid support through a covalent linkage,and wherein the density of the array of chemical compounds is at least1000 spots per cm².
 20. A method of identifying small molecule partnersfor biological macromolecules of interest, the method comprising:providing the array of claim 1, wherein the array comprises a pluralityof more than one type of chemical compound, and wherein the density ofthe array of chemical compounds is at least 1000 spots per cm²;contacting the array with one or more types of biological macromoleculesof interest; and determining the binding of specific chemicalcompound-biological macromolecule partners.